1. Field of the Invention
The present invention relates generally to particle analyzing apparatus and more particularly to apparatus and method for achieving selective, discriminate, differential classification of individual blood cell types, for example, five basic types of leukocytes, by means of technology of the Coulter type without the utilization of cytochemical staining techniques or materials.
2. Description of the Prior Art
It is well known that leukocytes can be discriminated and classified into three main types, lymphocytes, monocytes, and granulocytes, by means of flow cytometry instrumentation and without the use of cytochemical staining. One such method involves using low angle light scatter in combination with 90.degree. or high angle light scatter. Some methodologies use a lysing reagent to remove unwanted erythrocytes from a whole blood dilution; others rely on density or centrifugation techniques, such as, ficol, dextran, "buffycoat", etc. In addition, some methodologies use cytochemical staining in order to further subclassify lymphocyte subsets, as defined by immunological function.
PCT/US85/00868 to S.L. Ledis and H.R. Crews, assigned to Coulter Electronics, Inc., Hialeah, Florida, describes a system and techniques for producing what is characterized as a four population differential by utilizing a reagent system in such a way that the erythrocytes or red cells are effectively removed from the sample and, due to the lysing and if necessary fixing process, a granulocyte subpopulation, namely eosinophils, is separated out, in contrast to the earlier described techniques wherein only three populations are made visible or apparent, the eosinophils then being undistinguishable from the other subpopulations.
The prior art describes a method and means for obtaining a four-part leukocyte differential--lymphocytes, monocytes, neutrophils, and eosinophils--by cytochemically staining the cells to yield four distinct groups by the combination of low angle light scatter and absorption. Absorption is a component of axial light loss.
Also, in a technique in which electrical "opacity", as taught in U.S. Pat. No. 3,502,974, is plotted as a separate parameter for detecting certain cells or clusters of cells, the three populations are once again differentiated and fairly well defined and separated. However, in this instance, the granulocyte subpopulations of neutrophils, eosinophils, and basophils, are within the granulocyte cluster data, which hides or masks these three from differentiation.
The prior art literature, scientific papers and reports, illustrate, describe, and discuss the use of light scatter techniques at a variety of angular positions relative to the axis of the light beam being utilized to illuminate and interrogate the sample. However, the majority of the literature material so far available and considered restricts the light intersecting angles to either 0.degree. to 23.degree. or 90.degree., or both relative to the light axis.